Unveiling Aggregation-Induced Emission in Benzimidazole-Acrylonitriles for Fluorescent Live-Cell Imaging in HeLa Cells

Abstract

Cellular imaging is a pivotal strategy for unraveling complex biological processes, disease mechanisms, and drug responses, wherein benzimidazole-acrylonitriles emerge as promising yet underexplored fluorogenic scaffolds for advanced imaging and therapeutic applications. In the present study, four benzimidazole-acrylonitrile conjugates bearing nitrogen-rich heterocycles were synthesized and systematically investigated for their photophysical characteristics and aggregation-induced emission (AIE) behaviour. The synthetic strategy involves a stepwise condensation of o-phenylenediamine with ethyl cyanoacetate, followed by an L-proline-catalyzed condensation of the resulting intermediate with nitrogen-rich arylaldehydes. The molecular structures of the synthesized compounds were confirmed by 1 H and 13 C NMR spectroscopy, and high-resolution mass spectrometry (HRMS); the structure of compound 4 was further unambiguously established by single-crystal X-ray diffraction analysis. Optical characterization revealed distinct absorption and emission features attributable to intramolecular charge transfer (ICT) transitions, along with pronounced aggregation-induced emission (AIE) behavior in THF/water mixtures. The observed AIE characteristics were further supported by scanning electron microscopy (SEM) and dynamic light scattering (DLS), which revealed the formation of well-defined aggregated morphologies. Density functional theory (DFT) calculations provided complementary insights into the electronic structure and optical transitions. Remarkably, compound 1 exhibited efficient cytoplasmic localization in live HeLa cells, demonstrating its potential utility as a fluorescent bioimaging probe. Collectively, these findings establish benzimidazole-acrylonitrile conjugates as a new class of AIE-active luminogens with promising applications in precision bioimaging, tumor diagnostics, and theranostic platforms.